Summary: The expression of virulence factors in the bacterium Bordetella pertussis is regulated in an environmentally responsive fashion under the control of the two-component system encoded by the bvgAS locus. In recent years we have focused research efforts on understanding the mechanisms of gene regulation in this important human pathogen - specifically focussing on the genes encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Within the past year we have performed a detailed genetic and biochemical analysis of two regions of the fha promoter, the high affinity primary binding site, and the lower affinity secondary binding region. The primary binding site analysis has helped us to define critical bases involved in DNA recognition and binding by BvgA, and to begin to predict the presence of binding sites in unknown sequences. The secondary binding region analysis revealed that there are no critical bases for BvgA interaction and binding in the context of cooperative interactions with BvgA bound to the primary site. DNA sequence analysis of a large number of random substitutions within either of these regions which were able to restore promoter activity has extended and confirmed these predictions. Recently, powerful biochemical tools have been employed to determine the stoichiometry and configuration of BvgA molecules bound to the promoter DNA of the fha operon. BvgA was substituted at different positions with cysteine and subsequently derivatized with Fe-BABE, a chemical cleavage reagent. Use of these derivatized BvgA molecules has revealed the exact location and orientation of BvgA molecules bound to DNA. These results are consistent with structural studies of NarL, a related response regulator protein from E. coli. Application of the same FeBABE labelling and cleavage techniques to the C-terminal domain of the alpha subunit of RNA polymerase have revealed that BvgA interaction with this key regulatory domain involves a novel form of interaction not previously described for a transcriptional activator. Furthermore, extension of these techniques to other BvgA regulated promoters has allowed a more accurate description of BvgA than has previously been possible. Together, these studies have provided a quantum leap in our understanding of BvgA interaction at virulence gene promoters and have thus increased our understanding of the mechanisms of virulence gene activation in this important human pathogen.